Herbert Wiesinger-Mayr

Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition

BackgroundPathogen identification in clinical routine is based on the cultivation of microbes with subsequent morphological and physiological characterisation lasting at least 24 hours. However, early and accurate identification is a crucial requisite for fast and optimally targeted antimicrobial treatment. Molecular biology based techniques allow fast identification, however discrimination of very closely related species remains still difficult.ResultsA molecular approach is presented for the rapid identification of pathogens combining PCR amplification with microarray detection. The DNA chip comprises oligonucleotide capture probes for 25 different pathogens including Gram positive cocci, the most frequently encountered genera of Enterobacteriaceae, non-fermenter and clinical relevant Candida species. The observed detection limits varied from 10 cells (e.g. E. coli) to 105 cells (S. aureus) per mL artificially spiked blood. Thus the current low sensitivity for some species still represents a barrier for clinical application. Successful discrimination of closely related species was achieved by a signal pattern recognition approach based on the k-nearest-neighbour method. A prototype software providing this statistical evaluation was developed, allowing correct identification in 100 % of the cases at the genus and in 96.7 % at the species level (n = 241).ConclusionThe newly developed molecular assay can be carried out within 6 hours in a research laboratory from pathogen isolation to species identification. From our results we conclude that DNA microarrays can be a useful tool for rapid identification of closely related pathogens particularly when the protocols are adapted to the special clinical scenarios.

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10(1) to 10(2) bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.

Long target droplet polymerase chain reaction with a microfluidic device for high-throughput detection of pathogenic bacteria at clinical sensitivity.

In this article we present a long target droplet polymerase chain reaction (PCR) microsystem for the amplification of the 16S ribosomal RNA gene. It is used for detecting Gram-positive and Gram-negative pathogens at high-throughput and is optimised for downstream species identification. The miniaturised device consists of three heating plates for denaturation, annealing and extension arranged to form a triangular prism. Around this prism a fluoropolymeric tubing is coiled, which represents the reactor. The source DNA was thermally isolated from bacterial cells without any purification, which proved the robustness of the system. Long target sequences up to 1.3 kbp from Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa have successfully been amplified, which is crucial for the successive species classification with DNA microarrays at high accuracy. In addition to the kilobase amplicon, detection limits down to DNA concentrations equivalent to 102 bacterial cells per reaction were achieved, which qualifies the microfluidic device for clinical applications. PCR efficiency could be increased up to 2-fold and the total processing time was accelerated 3-fold in comparison to a conventional thermocycler. Besides this speed-up, the device operates in continuous mode with consecutive droplets, offering a maximal throughput of 80 samples per hour in a single reactor. Therefore we have overcome the trade-off between target length, sensitivity and throughput, existing in present literature. This qualifies the device for the application in species identification by PCR and microarray technology with high sample numbers. Moreover early diagnosis of infectious diseases can be implemented, allowing immediate species specific antibiotic treatment. Finally this can improve patient convalescence significantly.

Multiplex detection of antibiotic resistance genes using padlock probes

The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse β-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the β-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10(4) DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of β-lactamase genes in dilutions of 10(7) cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.

High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis.

The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 β-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europes largest hospitals, the General Hospital Vienna, were screened for the presence of 31 β-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of β-lactamase genes was estimated using biostatistical models. The carbapenemase genes blaKPC-2, blaKPC-3, and blaVIM-4 were identified in carbapenem-resistant and intermediate susceptible isolates, blaOXA-72 in an extended-spectrum β-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired blaDHA-1 and blaCMY AmpC β-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all β-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated β-lactamases and other resistance genes in one of Europes largest hospitals.

Human blood monocytes support persistence, but not replication of the intracellular pathogen C. pneumoniae

BackgroundIntracellular pathogens have devised various mechanisms to subvert the host immune response in order to survive and replicate in host cells. Here, we studied the infection of human blood monocytes with the intracellular pathogen C. pneumoniae and the effect on cytokine and chemokine profiles in comparison to stimulation with LPS.ResultsMonocytes purified from peripheral blood mononuclear cells by negative depletion were infected with C. pneumoniae. While immunofluorescence confirmed the presence of chlamydial lipopolysaccharide (LPS) in the cytoplasm of infected monocytes, real-time PCR did not provide evidence for replication of the intracellular pathogen. Complementary to PCR, C. pneumoniae infection was confirmed by an oligonucleotide DNA microarray for the detection of intracellular pathogens. Raman microspectroscopy revealed different molecular fingerprints for infected and non-infected monocytes, which were mainly due to changes in lipid and fatty acid content. Stimulation of monocytes with C. pneumoniae or with LPS induced similar profiles of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, but higher levels of IL-1β, IL-12p40 and IL-12p70 for C. pneumoniae which were statistically significant. C. pneumoniae also induced release of the chemokines MCP-1, MIP-1α and MIP-1β, and CXCL-8, which correlated with TNF-α secretion.ConclusionInfection of human blood monocytes with intracellular pathogens triggers altered cytokine and chemokine pattern as compared to stimulation with extracellular ligands such as LPS. Complementing conventional methods, an oligonucleotide DNA microarray for the detection of intracellular pathogens as well as Raman microspectroscopy provide useful tools to trace monocyte infection.

A multi-purpose ultrasonic streaming mixer for integrated magnetic bead ELISAs

We present an ultrasonic streaming mixer for disposable and on-chip magnetic bead ELISAs. The ultrasonic transducer is placed at system-level to keep cost per chip as low as possible, and is coupled to the chip by means of a solid ultrasonic horn. The system provides mixing of liquids, as well as dispersion of the superparamagnetic beads in the ELISA. Additionally it can be used clean the chamber surface from nonspecifically bound proteins during the washing steps in the ELISA protocol. Using our system the time for the ELISA protocol has been greatly reduced down to 30 min.

Removal of nonspecific bindings in on-chip ELISAs with low power ultrasound

We present a novel method for removing nonspecifically bound proteins for on-chip ELISAs (Enzyme-Linked-Immuno-Sorbent-Assays) by integrated ultrasonic washing of the reaction chamber. Low power ultrasound is concentrated into the reaction chamber with an ultrasonic horn contained in the base of the measurement system. Application of ultrasonication during the last washing step of the ELISA protocol removes adsorbed proteins from the wall while not affecting specifically bound analytes. This method reduces the cost of the disposable assay-chips, while delivering comparable results with standard blocking methods.

Hybridisation mix synthesis in a spiral lab-on-chip device for fast-track microarray genotyping of human pathogens

DNA microarrays can provide bacterial identification, which is crucial for targeted therapy. However they lack rapidness, because of multiple analysis steps. Therefore a fast one-step method for synthesising a hybridisation-ready reagent out of initial bacterial DNA is required. This work presents the combination and acceleration of PCR and fluorescent labelling within a disposable microfluidic chip, fabricated by injection moulding. The utilised geometry consists of a spiral meander with 40 turns, representing a cyclic-flow PCR system. The used reaction chemistry includes Cy3-conjugated primers and a high-yield polymerase leading to a one-step process accelerated by cyclic-flow PCR. Three different bacterial samples (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) were processed and the bacterial DNA was successfully amplified and labelled with detection limits down to 102 cells per reaction. The specificity of species identification was comparable to the approach of separate PCR and labelling. Furthermore the overall processing time was decreased from 6 hours to 1.5 hours. We showed that a disposable polycarbonate chip, fabricated by injection moulding is suitable for the significant acceleration of DNA microarray assays. The reaction output lead to high-sensitivity bacterial identification in a short time, which is crucial for an early and targeted therapy against infectious diseases.

Corrigendum to “High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis” [Infect. Genet. Evol., Oct.;27 (2014) 408–417]

The authors regret that the clinical isolate #C56 was wrongly classified as Escherichia coli. The isolate was initially characterised as Klebsiella pneumoniae in the hospital. Upon whole genome sequencing, analysis and annotation using the online tool RAST, the software indicated that the sequenced strain is most closely related to E.coli strains. Thus, the strain was reclassified as E. coli. However, after further more detailed reanalysis the strain #C56 has to be classified as K. pneumoniae. The authors would like to apologise for any inconvenience caused.